Pyrimidine nucleotide-sensitive phosphoinositidase C activity (PLC), previously identified in frog semicircular canal ampulla, was pharmacologically characterized. Binding of [³H]UTP and abilities of unlabeled nucleotide analogs to inhibit binding and to stimulate PLC in myo-[³H]inositol-loaded ampullas were determined. Specific [³H]UTP binding was competitively inhibited by UTP [apparent dissociation binding constant = 0.8 µM; Hill coefficient = 0.7]. Scatchard analysis revealed a minor class of high-affinity binding sites [45 fmol UTP bound/µg protein; dissociation constant (K_D1) = 0.4 µM] and a major class of moderate-affinity binding sites (365 fmol UTP bound/µg protein; K_D2 = 10 µM). The stereospecificity pattern for UTP analog recognition was UMP > UDP  ADP = UTP = dTTP > adenosine 5'-O-(3-thiotriphosphate) = ATP = CTP = 2'-and 3'-O-4-(benzoylbenzoyl)-ATP (Bz-ATP)  AMP  2-methylthio-ATP = ,-methylene-ATP > uridine = diadenosine tetraphosphate (Ap₄A); cAMP and adenosine were inactive. Antagonist recognition pattern was DIDS = pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) = reactive blue 2 > suramin. The rank order of potencies for agonist-induced PLC activation was UDP  UTP  Ap₄A  UMP = Bz-ATP; uridine was inactive. UTP-stimulated PLC activity was inhibited by DIDS = reactive blue 2 = PPADS > suramin. These results suggest that the population of [³H]UTP-labeled binding sites is heterogeneous, with a low number of high-affinity UTP receptors whose function(s) need to be determined and a large number of moderate-affinity receptors triggering PLC activation.